The human epidermal growth factor receptor-2 (HER2/neu; erbB2) oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor (EGFR). erbB2 belongs to a family of four transmembrane receptor tyrosine kinases involved in signal transduction pathways that regulate cell growth and proliferation in tissues of epithelial, mesenchymal and neuronal origin. Ligand binding to erbB2 receptors results in dimerization, including heterodimerization with other EGFR family members such as c-erbB3 and EGFR, and kinase activation, followed by phosphorylation of tyrosine residues in the intracellular receptor cytoplasmic tail. Phosphorylated tyrosines provide recognition sites for intracellular signaling intermediates that provide the link to downstream transduction cascades. Amplification or over-expression of erbB2 leads to transformation in the absence of a ligand through enhanced cell proliferation, motility and adhesion.
Clinical studies indicate that c-erbB2/HER2 is overexpressed in certain types of tumors of epithelial origin. Cancers that originate from epithelial cells, including those of the breast, lung, prostate, ovary, stomach, pancreas, bladder, rectum, colon, kidney, head and neck, as well as glioblastoma and adenocarcinoma, are by far the most common types of cancer in adults. Over-expression of erbB2 is present in about 30% of invasive human breast and ovarian cancers and is associated with a poor clinical outcome, including short survival time and short time to relapse. Similarly, c-erbB2 gene amplification or over-expression has been reported in ovarian cancer. c-erbB2 is also amplified and/or over-expressed in both benign and malignant prostate tissue, where c-erbB2 over-expression is associated with large tumor volume, high tumor grade and distant metastases, as well as in 50% of invasive bladder cancers. Development of adenocarcinoma in the esophagus and cancer of the stomach have also been associated with over-expression of the c-erbB2.
Because of the prognostic and predictive value of c-erbB2, the status of c-erbB2 is routinely tested in invasive cancers. Methodologies to identify over-expression of HER2 include immunohistochemistry (IHC); silver, chromogenic, or fluorescent in situ hybridization (SISH/CISH/FISH); and PCR-based technologies.
HER-2 protein over-expression is routinely determined by IHC. Highly standardized, semi-quantitative IHC assays and scoring procedures have been developed which categorize HER2 expression levels in a scale from 0 to 3+, with 0 being lack of protein expression and 3+ corresponding to cells containing approximately 2,300,000 receptors/cell, showing HER2 over-expression in more than 10% of the cells. Advantages of IHC testing include its wide availability, relatively low cost, easy preservation of stained slides, and use of a familiar routine microscope. Disadvantages include the impact of pre-analytic issues, including, for example, storage, duration and type of fixation, type of antibody, nature of system control samples, and, importantly, the difficulties in applying a subjective slide scoring system. Fluorescent in-situ hybridization (FISH) determines the level of c-erbB2 over-expression in patients, by detecting gene amplification. This technique is expensive and requires a fluorescent microscope and image captures system. Currently, the recommended assays most commonly used for determining the c-erbB2/HER2 status of breast cancer tissue are a combination of IHC and FISH, where tissue samples receiving IHC scores of 0 and 1+ are considered negative for HER2 over-expression, and those receiving scores of 3+ are positive for HER2 over-expression. Tissue samples receiving scores of 2+ and 2+/3+ are reexamined using FISH for a final determination.
HER2/neu over-expression is often used to predict which cancer patients are most likely to benefit from certain cancer treatments that directly bind with the HER2/neu protein and modulate its biological activity via a number of mechanisms. Thus, trastuzumab, which is marketed under the tradename HERCEPTIN® by Genentech Corporation, South San Francisco, Calif., is an FDA-approved drug for use in HER2-positive metastatic breast cancer in combination with paclitaxel as a first-line therapy, and as a single agent in second- and third-line therapy for metastatic breast cancer patients after other therapies have failed. HERCEPTIN® has also been approved by the FDA for the adjuvant treatment of patients with HER-2 positive node-positive breast cancer as part of a treatment regimen containing doxorubicin, cyclophosphamide and paclitaxel, and as a single agent for the adjuvant treatment of HER-2 over-expressing node-negative or node-positive breast cancer, following multi-modality anthracycline-based therapy. Trastuzumab is a humanized mouse monoclonal antibody that specifically binds the C-terminal end of domain IV of the extracellular region of the HER2/neu receptor. Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle, leading to a reduction in cell proliferation. Trastuzumab is believed to induce some of its effects by down-regulating HER2/neu, leading to the disruption of receptor dimerization and cell signaling through downstream signaling cascades.
Trastuzumab therapy was initially targeted specifically for patients with advanced relapsed breast cancer that over-expressed the HER2/neu protein. Currently, initiation of trastuzumab therapy is based upon the identification of HER2/neu over-expression in the breast tumor tissue of a patient, under the assumption that those patients most likely to respond to trastuzumab therapy are those having breast cancer that over-expresses HER2/neu+ (“HER2/neu+ patients”).
Lapatinib, marketed by GlaxoSmithKline (GSK) under the trade names TYKERB® and TYVERB®, is an ATP-competitive epidermal growth factor receptor (EGFR) and HER2/neu (ErbB-2) dual tyrosine kinase inhibitor, that inhibits receptor auto-phosphorylation and activation by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain. Lapatinib has shown remarkable activity both in vitro and in vivo, leading to growth arrest and apoptosis of tumor cell lines that over-express EGFR or HER2 in a variety of tumors, including breast and renal cancers. Lapatinib is approved by the FDA for use in patients with advanced metastatic breast cancer in conjunction with the chemotherapy drug capecitabine and is indicated for patients with resistance to trastuzumab.
In view of the utility of HER2 as a predictive marker, the American Society of Clinical Oncology and the College of American Pathologists convened an expert panel that developed recommendations for optimal HER2 testing performance and recommended that HER2 status should be determined for all invasive breast cancer.
Despite the commonly-accepted expectation that HER2 amplification in cancer patients predicts benefit from trastuzumab therapy for these patients, a recent publication from Paik et al. (NEJM, Mar. 27, 2008), found no significant association between HER2 gene copy number and patient benefit, and provided strong evidence that approximately 10% of the HER2-negative patients studied in a large cohort of a National Surgical Adjuvant Breast and Bowel Project (NSABP) trial responded to and benefited from trastuzumab therapy. Moreover, therapeutic success, as defined by statistical significance in overall survival, or progression free survival or time to progression, for breast cancer patients with high expression of c-erbB2 (e.g. FISH+ and/or IHC 3+) is only approximately 50%. In other words, half of the patients with high expressing HER2 protein do not respond to HER2-directed therapy and about 10% of HER2-patients respond to therapy despite having low levels of the protein expressed.
Accordingly, there is an urgent need in the art to develop a more reliable and accurate method(s) to identify subjects with a cancer of epithelial origin who would respond to and benefit from trastuzumab therapy or treatment with a c-erbB2 kinase inhibitor, for selection and inclusion for erbB2-directed treatment and therapy. The present invention satisfies this need.